**2. The contamination problem**

*Aspergillus parasiticus* often grows in oily products such as peanuts, walnuts, pistachios, pine nuts, pumpkin seeds and sunflower seeds, while *A. flavus* is commonly found contaminating agricultural fields of grains such as corn, sorghum, rice, barley, rye, and oats, as well as in spices (chili, pepper, mustard, and cloves). All of these commodities or products may be raw material for animal feed, which when ingested may pass into breast milk and can later be found in cheese, yogurt, cream, meat, and egg, constituting a source of secondary contamination for humans (Juan-López et al., 1995).

Fungal invasion and aflatoxin contamination often begin before harvest and can be promoted by production and harvest conditions, genotypes, drought, soil types, and insect activity, among other factors (Cole et al., 1995; Lynch & Wilson, 1991; Mehan et al., 1986, 1991). Therefore, timely harvest and rapid and adequate drying before storage are important factors to avoid or reduce post-harvest contamination, because even moisture generated by insect respiration and local condensation may develop local pockets favorable to aflatoxin growth (Mehan et al., 1986; Williams et al., 2004). This may partially explain differences in the range of contaminated products among countries. For example, in Japan aflatoxins were detected in about 50% of peanut butter and bitter chocolate samples, while their presence was not found in corn products; in contrast, a study in China reported contamination in 70% of corn products (Kumagai et al., 2008; Wang & Liu, 2007).

Aflatoxin contamination may be more severe in developing than in developed countries, yet this is a worldwide problem that could reach as much as 25% of the world's crops (Fink-Gremmels, 1999). In past years, a survey conducted in Midwestern states of the USA found 19.5% of corn samples contaminated with aflatoxin when assayed prior to any induced environmental stress, and 24.7% of them contaminated following stress induction (Russell et al., 1991). Also, Shane (1993) estimated losses in Southeastern USA for around 97 million dollars because of AFB1-contaminated corn with an additional 100 million dollars in production losses at hog farms feeding the contaminated grain.


Table 1. Taxonomy of *Aspergillus*

Their spores are produced in large numbers and are spread widely by air currents. These molds grow within many commodities when temperatures are between 24-35 °C, and the

The present chapter has the purpose of putting into perspective the worldwide relevance of the AFB1 contamination problem due to its effect on the aspects of economy and health, as well as to review the main strategies developed for coping with such contamination. In particular, we discuss the theoretical grounds and the practical approaches which have been carried out by using antimutagenesis and chemoprevention strategies. In these areas are included a description and discussion of the more relevant agents tested against the

*Aspergillus parasiticus* often grows in oily products such as peanuts, walnuts, pistachios, pine nuts, pumpkin seeds and sunflower seeds, while *A. flavus* is commonly found contaminating agricultural fields of grains such as corn, sorghum, rice, barley, rye, and oats, as well as in spices (chili, pepper, mustard, and cloves). All of these commodities or products may be raw material for animal feed, which when ingested may pass into breast milk and can later be found in cheese, yogurt, cream, meat, and egg, constituting a source of

Fungal invasion and aflatoxin contamination often begin before harvest and can be promoted by production and harvest conditions, genotypes, drought, soil types, and insect activity, among other factors (Cole et al., 1995; Lynch & Wilson, 1991; Mehan et al., 1986, 1991). Therefore, timely harvest and rapid and adequate drying before storage are important factors to avoid or reduce post-harvest contamination, because even moisture generated by insect respiration and local condensation may develop local pockets favorable to aflatoxin growth (Mehan et al., 1986; Williams et al., 2004). This may partially explain differences in the range of contaminated products among countries. For example, in Japan aflatoxins were detected in about 50% of peanut butter and bitter chocolate samples, while their presence was not found in corn products; in contrast, a study in China reported contamination in 70%

Aflatoxin contamination may be more severe in developing than in developed countries, yet this is a worldwide problem that could reach as much as 25% of the world's crops (Fink-Gremmels, 1999). In past years, a survey conducted in Midwestern states of the USA found 19.5% of corn samples contaminated with aflatoxin when assayed prior to any induced environmental stress, and 24.7% of them contaminated following stress induction (Russell et al., 1991). Also, Shane (1993) estimated losses in Southeastern USA for around 97 million dollars because of AFB1-contaminated corn with an additional 100 million dollars in

**Kingdom Fungi** 

moisture content exceeds 7% -10% (Kogbo et al., 1985; Williams et al., 2004).

genotoxic and carcinogenic damage induced by AFB1.

secondary contamination for humans (Juan-López et al., 1995).

of corn products (Kumagai et al., 2008; Wang & Liu, 2007).

production losses at hog farms feeding the contaminated grain.

Phylum Ascomycota Class Eurotiomycetes Order Eurotiales Family Trichocomaceae Genus *Aspergillus* 

Table 1. Taxonomy of *Aspergillus*

**2. The contamination problem** 

There are diverse criteria for assessing the economic impact of aflatoxins. These include loss of human and animal life, health care and veterinary care costs, loss of livestock production, loss of forage crops and feeds, regulatory costs, and research cost focusing on relieving the impact and severity of the aflatoxin problem. However, most reports on the matter are on a single aspect of aflatoxin exposure or contamination.

With regard to the heavy impact of AFB1 contamination, India can be an example of the problem in emerging countries. A study in the Bihar region showed that nearly 51% of the 387 samples tested were contaminated with molds, and that from the 139 samples containing AFB1, 133 had levels above 0.02 mg/kg (Ranjan & Sinha, 1991). In other studies, authors found levels as high as 3.7 mg/kg of AFB1 in groundnut meal used for dairy cattle, as well as 0.05 to 0.4 mg/kg in 21 of 28 dairy feed samples from farms in and around Ludhiana and Punjab (Dhand et al., 1998; Phillips et al., 1996). Also, in raw peanut oil 65-70 % of AFB1 was found in the sediment and 30-35 % in the supernatant oil after centrifugation (Banu & Muthumary, 2010a). In this context, groundnut contamination was estimated to represent about a 10 million dollar loss in India's export within a decade (Hussein & Brasel, 2001; Vasanthi & Bhat, 1998). Regarding the extent of the problem in developing countries, Table 2 shows that a wide range of commodities are contaminated, even to a higher degree than usually allowed (Williams et al., 2004).

In Mexico the main contaminated crop is corn. This is a logical situation considering that the country has one of the highest rates of human consumption of this grain in the world (120 kg per capita per year) with a production of about 10.2 million tons for human consumption and 5 million tons for animal feed and other industries (Plasencia, 2004). One of the most significant episodes of aflatoxin contamination of maize was probably that which occurred in a northern state (Tamaulipas) in 1989, where levels of the toxin above 0.1 mg/kg were reported in practically all the plants harvested (García & Heredia, 2006). This represents a potential high health risk to the population, because corn is a basic food consumed as tortilla, with a consumption of 325 g per day (Anguiano-Ruvalcaba et al., 2005). However, this is not the only food susceptible to AFB1 that may pose a health risk, because a number of other maize-based foods are part of the Mexican diet. In regard to this contamination a few studies have been made. In kernelled corn for human consumption in the city of Monterrey, AFB1 was determined in 36 of the 41 samples tested, with concentrations ranging from 5 to 465 ng/g, with 59% of those samples above the Mexican legal limit of 0.02 mg/kg (Torres-Espinoza et al., 1995). Another study in 66 stored samples of maize and wheat in the state of Sonora showed 13 samples (20%) contaminated with AFB1, although the level was higher than 0.02 mg/kg in only one sample (Ochoa et al., 1989). Some general explanations for the contamination in the country are the following: 1- inadequate pre-harvest and storage management, as well as distribution procedures that may favor the development of *Aspergillus*; 2- corn growing under non-irrigation conditions in many places, predisposing plants to drought stress and mold infection; 3- limited possibilities of modern agricultural practices for low income farmers; 4- legal restriction for the use of transgenic maize manifesting insecticidal proteins or any other trait to reduce aflatoxin contamination; 5-infestation with the microleopterans *Carpophilus freemani*, the sap beetle, *Sitophilus zeamais*, the maize weevil, and *Cathartus quadricollis*, square-necked grain beetle, which may facilitate spore entry in the cobs; 6- growth of pollinated varieties which appear to be more prone to disease development and to the effect of environmental factors in comparison with maize hybrids (Figueroa, 1999; Plasencia, 2004; Zuber et al., 1983).

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 255

AFB1 was first isolated some 40 years ago after outbreaks of disease and death occurred in turkeys and rainbow trout fed on contaminated peanut and cottonseed meals (Williams et al., 2004). From this time onwards a number of investigations have corroborated the strong toxicity of this mycotoxin in mammals, poultry, fish and other animals (Girish & Smith, 2008; Kensler et al., 2011; Madrigal-Santillán et al., 2010; Santacroce et al., 2008). Aflatoxicosis is the poisoning that results from ingesting AFB1, and two general forms of the affection have been identified. One is an acute, severe intoxication, which results in direct liver damage and subsequent illness or death, related with large doses; this type of aflatoxicosis includes symptoms such as hemorrhagic necrosis of the liver, bile duct proliferation, edema, lethargy, and liver cirrhosis The other, a chronic form of the disease, corresponds to a subsymptomatic exposure, which is related with nutritional and immunologic consequences, such as suppression of the cell-mediated immune responses; also, as dose exposure has a cumulative effect, there can be a significant risk increase of

Studies on the matter have established a species-related susceptibility to health effect by AFB1, and a role of the dose and the duration of the exposure (Neiger et al., 1994; Pestka & Bondy, 1994; Silvotti et al., 1997); nevertheless, it has been clearly shown that AFB1 is a powerful carcinogen for humans and many animal species, including rodents, non-human primates, and fish (Kimura et al., 2004; Santacroce et al., 2008). The main target of the agent is the liver, although tumors may also develop in other organs, such as the lungs, kidney and colon (Wang & Groopman, 1999). Therefore since 1993, The International Agency for Research on Cancer (IARC) has classified it as a high potential carcinogenic agent (Class I) (IARC, 1993). Besides, a strong synergy between aflatoxin and the presence of hepatitis B and C viruses has also been determined, a combination that significantly increases the risk for having liver cancer, as shown in places like Gambia, and Qidong, China (Wang et al.,

Fig. 2. Biotransformation pathways of aflatoxin B1

developing cancer (Steyn, 1995; Williams et al., 2004).

1996, 2001).

**3. Toxicity and intervention strategies** 


Table 2. Examples of market sample contamination, frequencies, and concentrations

Besides economical and educational actions that can be carried out to reduce the contamination problem in Mexico, other specific actions can be the following: 1- more research and breeding programs to identify varieties resistant to fungal infection and AFB1 contamination; 2- epidemiological data concerning liver cancer/AFB1 ingestion, as well as determination of AFB1 intake and its excretion in fluids, (particularly because cancer initiation may take about 6 years); 3- adoption of a standard method for measuring AFB1 content at both national and international levels, which must be sensitive, reliable, reproducible, and cost-effective.

Fig. 2. Biotransformation pathways of aflatoxin B1

#### **3. Toxicity and intervention strategies**

254 Aflatoxins – Detection, Measurement And Control

Maize (95) 67 33.0 (mean)

Corn (99) 76 >20.0

Maize (100) 80 >20.0

Incaparina (corn/ cottonseed flour) (106) 100 3.0-214.0

Wheat (117) 1.2 >25.62

Corn (118) 87.8 5.0-465.0

Pistachio (121) 8.7 to 33 >20.0

Peanut oil (122) 85 40.0 (mean) Table 2. Examples of market sample contamination, frequencies, and concentrations

Besides economical and educational actions that can be carried out to reduce the contamination problem in Mexico, other specific actions can be the following: 1- more research and breeding programs to identify varieties resistant to fungal infection and AFB1 contamination; 2- epidemiological data concerning liver cancer/AFB1 ingestion, as well as determination of AFB1 intake and its excretion in fluids, (particularly because cancer initiation may take about 6 years); 3- adoption of a standard method for measuring AFB1 content at both national and international levels, which must be sensitive, reliable,

samples (%)

38.3 67

56.7 90 35

> 18 26

> 12 19

> 45 25

Contamination rate (ppb)

> 0.2-129.0 43.0-1099.0

>10.0 25.0-175.0 5.0-35.0

> >30.0 >30.0

26.0 (mean) 74.0

25.0-770.0 0.002-19.716

Country/ commodity/Number Positive AFB1

*Bangladesh* 

*Brazil*  Corn (96) Peanut (97)

*China* 

*Egypt* 

*Costa Rica* 

*Guatemala* 

Chilies (109) Maize (113)

Barley food (114) Corn food (114)

Maize-based gruels (120)

reproducible, and cost-effective.

*India* 

*Korea* 

*Malaysia* 

*Mexico* 

*Nigeria*  Corn (119)

*Qatar* 

*Senegal* 

Peanut butter (101) Hazelnut (102) Soybean (104)

> AFB1 was first isolated some 40 years ago after outbreaks of disease and death occurred in turkeys and rainbow trout fed on contaminated peanut and cottonseed meals (Williams et al., 2004). From this time onwards a number of investigations have corroborated the strong toxicity of this mycotoxin in mammals, poultry, fish and other animals (Girish & Smith, 2008; Kensler et al., 2011; Madrigal-Santillán et al., 2010; Santacroce et al., 2008). Aflatoxicosis is the poisoning that results from ingesting AFB1, and two general forms of the affection have been identified. One is an acute, severe intoxication, which results in direct liver damage and subsequent illness or death, related with large doses; this type of aflatoxicosis includes symptoms such as hemorrhagic necrosis of the liver, bile duct proliferation, edema, lethargy, and liver cirrhosis The other, a chronic form of the disease, corresponds to a subsymptomatic exposure, which is related with nutritional and immunologic consequences, such as suppression of the cell-mediated immune responses; also, as dose exposure has a cumulative effect, there can be a significant risk increase of developing cancer (Steyn, 1995; Williams et al., 2004).

> Studies on the matter have established a species-related susceptibility to health effect by AFB1, and a role of the dose and the duration of the exposure (Neiger et al., 1994; Pestka & Bondy, 1994; Silvotti et al., 1997); nevertheless, it has been clearly shown that AFB1 is a powerful carcinogen for humans and many animal species, including rodents, non-human primates, and fish (Kimura et al., 2004; Santacroce et al., 2008). The main target of the agent is the liver, although tumors may also develop in other organs, such as the lungs, kidney and colon (Wang & Groopman, 1999). Therefore since 1993, The International Agency for Research on Cancer (IARC) has classified it as a high potential carcinogenic agent (Class I) (IARC, 1993). Besides, a strong synergy between aflatoxin and the presence of hepatitis B and C viruses has also been determined, a combination that significantly increases the risk for having liver cancer, as shown in places like Gambia, and Qidong, China (Wang et al., 1996, 2001).

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 257

Actions to fulfill regulations or to correct possible failures can be taken at the phases of production, storage, and processing. At the initial steps, insect control can be performed, and improvements made in irrigation practices and storage structures as well as in the inoculation of non-aflatoxigenic strains; in the latter step, the actions can refer to the separation of the contaminated product, its dilution with grains lacking AFB1, or its decontamination through a number of physical and chemical methods which are designed to degrade, destruct, inactivate or remove the toxin. The ideal decontamination procedure should be easy to use and inexpensive, and it should not lead to the formation of compounds that are still toxic, or that may reverse to compounds that reform the parent mycotoxin or alter the nutritional and palatability properties of the grain or grain products. This has been a difficult task, and thus a number of methods have been proposed, showing variable results. Examples of these methods are presented in Table 3 (Madrigal-Santillán et al., 2010). The widespread contamination of AFB1, in addition to the complexity and danger of its toxicity, has suggested that not only one form of control and prevention can cope with the problem; this is a conviction that has promoted the development of different strategies. One of these refers to the application of antimutagenesis and chemoprevention procedures, as the basis to avoid or reduce DNA lesions, as well as other molecular and cellular alterations related with the process of cancer initiation. These studies can be carried out by inhibiting the formation of active AFB1 metabolites, avoiding the interaction with target macromolecules, or by accelerating the detoxication and repair processes, among other mechanisms. Comparison of antimutagenic or chemopreventive activities with biochemical and organic quantifications are relevant to confirm the efficacy of the prevention strategy.

**Physical methods Specific examples** 

Inactivation by radiation Ultraviolet light

Extraction by organic solvents Ethanol 95%

The relationship between chemical exposure and cancer development was observed about 140 years ago when an increase was noted in the cancer mortality rate of workers

Vapor pressure Microwave treatment Nixtamalization

Gamma radiation

Zeolites Bentonites Aluminosilicates

Acetone 90%

Hexane-ethanol Hydrogen peroxide Ammonium hydroxyde Methylamine Sodium hypochlorite

Inactivation by heat

Elimination by adsorbents

**Chemical methods** 

Chemical destruction

Table 3. AFB1 decontamination procedures

**4. Antimutagenesis and chemoprevention** 

AFB1 is absorbed in the small intestine and distributed by the blood throughout the body. Examination of the physicochemical and biochemical characteristics of the AFB1 molecule has revealed two important sites for toxicological activity. One is the double bond in position C / C-8,9, of the furo-furan ring. The aflatoxin-DNA and protein interaction at this site can alter the functioning of these macromolecules leading to cellular deleterious effects. Another reactive group is the lactone ring in the coumarin moiety, which is easily hydrolyzed and therefore, vulnerable for degradation (Banu & Muthumary, 2010b). AFB1 is metabolically activated by cytochrome P450 enzymes to yield two chemically reactive epoxides: AFB1-8,9-*exo* and -8,9-*endo* epoxides (Figure 2). However, only the 8,9-*exo* isomer reacts readily with DNA, forming the N7-guanine and its derivative AFB1-formamidopyrimidine adduct (Johnson & Guengerich, 1997). These events constitute the basis of AFB1 genotoxicity, which includes promutagenic and mutagenic events that can result in the activation of protooncogenes and the inactivation or loss of tumor suppressor genes. The formed epoxide is very unstable in water but can be handled relatively easily in aprotic solvents. CYP enzymes, on the other hand, also oxidize AFB1 to deactivated products that are generally poor substrates for epoxidation, or to those which after that step do not interact with DNA, including AFM, AFQ, and the *endo*-epoxide (Johnson & Guengerich, 1997; Guengerich et al., 1998).

The genotoxic effects induced by AFB1 have been extensively documented. The chemical is known to inhibit DNA synthesis, as well as DNA-dependent RNA polymerase activity messenger RNA synthesis, and protein synthesis (McLean & Dutton, 1995; Wang & Groopman, 1999). Furthermore, its strong genotoxicity has been demonstrated in many endpoints and model systems which include HeLa cells, *Bacillus subtillis, Neurospora crassa, Salmonella typhimurium,* CHO cells, chromosomal aberrations, sister chromatid exchanges (SCE), micronucleus, unscheduled DNA synthesis, DNA strand breaks, and DNA adducts (Anwar et al., 1994; El-Zawahri et al., 1990; Le Hegarat et al., 2010; Miranda et al., 2007; Theumer et al., 2010).

The above mentioned genotoxicity is in line with the induction of cancer by aflatoxins. Hepatocellular carcinoma is one of the most common malignancies worldwide, and a major risk factor includes dietary exposure to AFB1. Genetic and epigenetic changes are involved in the pathogenesis of the disease, including G:C to T:A transversions at the third base of codon 249 of the tumor suppressor gene *p53*. Besides, chronic infection with hepatitis virus, and the generation of reactive oxygen/nitrogen species can also damage DNA and mutate cancer-related genes, such as *p53*. One of the functions of this gene is to regulate the transcription of protective antioxidant genes, however, when the DNA is damaged, *p53* regulates the transcription of protective antioxidant genes, but with extensive DNA damage it transactivates pro-oxidant genes that contribute to apoptosis. Also, genes from the hepatitis B virus can be integrated in the genome of hepatocellular carcinoma cells, and mutant proteins may still bind to p53 and attenuate DNA repair and apoptosis; thus, it is clear that viruses and chemicals may be involved in the etiology of mutations during the molecular pathogenesis of liver carcinoma (Hussain et al., 2007; Oyaqbemi et al., 2010).

The strong toxicity of AFB1, which may be reflected in financial and social problems, prompted countries to incorporate regulations concerning the levels of mycotoxins in food and feed. In the case of AFB1, the maximum tolerated level varies from 1 to 20 μg/kg. The limit of 4 μg/kg is usually applied in countries that follow the harmonized regulations of the European Free Trade Association (EFTA) and the European Union (EU), and the 20 μg/kg limit is mainly applied in Latin American countries, the United States, and Africa (Guzmán de la Peña & Peña Cabrales, 2005).

AFB1 is absorbed in the small intestine and distributed by the blood throughout the body. Examination of the physicochemical and biochemical characteristics of the AFB1 molecule has revealed two important sites for toxicological activity. One is the double bond in position C / C-8,9, of the furo-furan ring. The aflatoxin-DNA and protein interaction at this site can alter the functioning of these macromolecules leading to cellular deleterious effects. Another reactive group is the lactone ring in the coumarin moiety, which is easily hydrolyzed and therefore, vulnerable for degradation (Banu & Muthumary, 2010b). AFB1 is metabolically activated by cytochrome P450 enzymes to yield two chemically reactive epoxides: AFB1-8,9-*exo* and -8,9-*endo* epoxides (Figure 2). However, only the 8,9-*exo* isomer reacts readily with DNA, forming the N7-guanine and its derivative AFB1-formamidopyrimidine adduct (Johnson & Guengerich, 1997). These events constitute the basis of AFB1 genotoxicity, which includes promutagenic and mutagenic events that can result in the activation of protooncogenes and the inactivation or loss of tumor suppressor genes. The formed epoxide is very unstable in water but can be handled relatively easily in aprotic solvents. CYP enzymes, on the other hand, also oxidize AFB1 to deactivated products that are generally poor substrates for epoxidation, or to those which after that step do not interact with DNA, including AFM, AFQ,

The genotoxic effects induced by AFB1 have been extensively documented. The chemical is known to inhibit DNA synthesis, as well as DNA-dependent RNA polymerase activity messenger RNA synthesis, and protein synthesis (McLean & Dutton, 1995; Wang & Groopman, 1999). Furthermore, its strong genotoxicity has been demonstrated in many endpoints and model systems which include HeLa cells, *Bacillus subtillis, Neurospora crassa, Salmonella typhimurium,* CHO cells, chromosomal aberrations, sister chromatid exchanges (SCE), micronucleus, unscheduled DNA synthesis, DNA strand breaks, and DNA adducts (Anwar et al., 1994; El-Zawahri et al., 1990; Le Hegarat et al., 2010; Miranda et al., 2007;

The above mentioned genotoxicity is in line with the induction of cancer by aflatoxins. Hepatocellular carcinoma is one of the most common malignancies worldwide, and a major risk factor includes dietary exposure to AFB1. Genetic and epigenetic changes are involved in the pathogenesis of the disease, including G:C to T:A transversions at the third base of codon 249 of the tumor suppressor gene *p53*. Besides, chronic infection with hepatitis virus, and the generation of reactive oxygen/nitrogen species can also damage DNA and mutate cancer-related genes, such as *p53*. One of the functions of this gene is to regulate the transcription of protective antioxidant genes, however, when the DNA is damaged, *p53* regulates the transcription of protective antioxidant genes, but with extensive DNA damage it transactivates pro-oxidant genes that contribute to apoptosis. Also, genes from the hepatitis B virus can be integrated in the genome of hepatocellular carcinoma cells, and mutant proteins may still bind to p53 and attenuate DNA repair and apoptosis; thus, it is clear that viruses and chemicals may be involved in the etiology of mutations during the molecular pathogenesis of liver carcinoma (Hussain et al., 2007; Oyaqbemi et al., 2010). The strong toxicity of AFB1, which may be reflected in financial and social problems, prompted countries to incorporate regulations concerning the levels of mycotoxins in food and feed. In the case of AFB1, the maximum tolerated level varies from 1 to 20 μg/kg. The limit of 4 μg/kg is usually applied in countries that follow the harmonized regulations of the European Free Trade Association (EFTA) and the European Union (EU), and the 20 μg/kg limit is mainly applied in Latin American countries, the United States, and Africa

and the *endo*-epoxide (Johnson & Guengerich, 1997; Guengerich et al., 1998).

Theumer et al., 2010).

(Guzmán de la Peña & Peña Cabrales, 2005).

Actions to fulfill regulations or to correct possible failures can be taken at the phases of production, storage, and processing. At the initial steps, insect control can be performed, and improvements made in irrigation practices and storage structures as well as in the inoculation of non-aflatoxigenic strains; in the latter step, the actions can refer to the separation of the contaminated product, its dilution with grains lacking AFB1, or its decontamination through a number of physical and chemical methods which are designed to degrade, destruct, inactivate or remove the toxin. The ideal decontamination procedure should be easy to use and inexpensive, and it should not lead to the formation of compounds that are still toxic, or that may reverse to compounds that reform the parent mycotoxin or alter the nutritional and palatability properties of the grain or grain products. This has been a difficult task, and thus a number of methods have been proposed, showing variable results. Examples of these methods are presented in Table 3 (Madrigal-Santillán et al., 2010). The widespread contamination of AFB1, in addition to the complexity and danger of its toxicity, has suggested that not only one form of control and prevention can cope with the problem; this is a conviction that has promoted the development of different strategies. One of these refers to the application of antimutagenesis and chemoprevention procedures, as the basis to avoid or reduce DNA lesions, as well as other molecular and cellular alterations related with the process of cancer initiation. These studies can be carried out by inhibiting the formation of active AFB1 metabolites, avoiding the interaction with target macromolecules, or by accelerating the detoxication and repair processes, among other mechanisms. Comparison of antimutagenic or chemopreventive activities with biochemical and organic quantifications are relevant to confirm the efficacy of the prevention strategy.


Table 3. AFB1 decontamination procedures

#### **4. Antimutagenesis and chemoprevention**

The relationship between chemical exposure and cancer development was observed about 140 years ago when an increase was noted in the cancer mortality rate of workers

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 259

activation at the cytochrome P-450. The other class of antimutagens corresponds to the bioantimutagens agents which may even reduce the level of mutations after the DNA has been damaged. To this group belong sequesters of mutagens and free radicals, agents that enhance the activity of phase II enzymes and the repair system, or those that reduce errors

The term chemoprevention was coined by Sporn in the mid 70s, and during the following years it was defined as a procedure for the prevention, inhibition, delay, or reversal of carcinogenesis by means of a variety of agents which include different nutrients, extracts of plants or pharmacologic compounds, among others. The aim of the strategy refers to finding agents with several characteristics: 1-low cost, as related to cost-benefit analyses and to the size of the target population; 2- practicality of use, regarding availability, storage conditions and administration route, besides taking into account the need to be used for long periods of time; 3-efficacy; and 4-safety. The selected chemopreventive agents should protect target molecules, cells, general population and individuals at risk, against the initiation, promotion or progression phases of carcinogenesis (De Flora & Ferguson, 2005). The concept is based in that chronic diseases may have common pathogenic determinants, such as DNA damage, oxidative stress, and chronic inflammation, and that a number of agents have proved to be efficient in blocking such alterations and in improving the quality and span of human life. The more promising candidates are subjected to clinical trials, which should be designed and conducted properly, and should include well characterized agents, suitable cohorts, and reliable biomarkers for measuring efficacy, which can serve as surrogate endpoints for cancer incidence. Phase II chemoprevention trials test promising agents for biomarkers modulation in cohorts of 30 to 200 participants at greater than average risk of the cancer under study; in contrast, phase III trials test agents for their efficacy in cancer prevention in thousands of participants who are generally healthy or who may be at slightly elevated risk

Genotoxicity/antigenotoxicity tests can be defined as in vitro and in vivo assays designed to detect both, compounds that induce genetic damage by various mechanisms, as well as those that prevent such damage. These tests enable hazard identification with respect to DNA damage and its fixation in the form of gene mutations, chromosomal aberrations or other alterations, all of which are considered essential for heritable effects and for the multistep process of malignancy (Figure 3). In contrast, the same tests may provide information on the level of protection and the mechanism involved regarding the antigenotoxic agents (Food and Drug Administration [FDA], 2008). In this section we will briefly describe the more basic fundaments regarding some of the tests most used to evaluate the prevention of

a. The Ames mutation assay, which was developed in *Salmonella typhimurium* in the mid 70s, is based in the use of strains with a mutation in the histidine locus which does not allow the bacteria to synthesize such aminoacid; thereby, reversion to the normal situation constitutes the mutagenic endpoint. The sensitivity of the test has been improved by incorporating mutations to the test organism, making it more permeable to chemicals and more resistant to DNA repair (Dearfield & Moore, 2005). Moreover, several strains which detect different base-pair substitutions have been constructed, thereby allowing the detection of oxidative damage or DNA cross-linking. Besides,

at the DNA replication level (Kada & Shimoi, 1987).

(Kelloff et al., 1995; Richmond & O´Mara, 2010).

**5. Genotoxicity/antigenotoxicity tests** 

DNA damage induced by AFB1.

managing coal tar. This effect was experimentally confirmed by Yamagiwa and Ichikawa (Weisburger, 2001) in exposed rabbits. Later, with the identification of the DNA structure and the development of new analytical methods, carcinogenesis was clearly shown to be related with alterations in this molecule. Such a relation was confirmed in the 60s, determining the effect exerted by metabolites of specific carcinogens on the structure and function of DNA and studying the activity of carcinogens on various cellular systems. In this context, the term genotoxic was then used for identifying carcinogens acting on DNA, in contrast with others whose effect was exerted through other routes (Weisburger, 2001). At this time, it was evident that the presence of numerous physical, chemical, and biological genotoxic agents could affect human health, and also that these genotoxicants may be present in a wide range of human activities, such as those related with work, food, health or personal habits. This produced a sort of genotoxic saturation, a condition which stimulated a search for knowledge about the agent's molecular, cellular, and metabolic peculiarities as well as detailed characteristics of the xenobiotic-DNA interaction; furthermore, the consequences of such an interaction were investigated, particularly with reference to human health.

A number of educational recommendations were then suggested in order to counteract the genotoxic effects, in addition to establishing regulatory measures related to permissible limits for specific substances; besides, efforts were made for substituting genotoxic drugs with less dangerous ones, including appropriate modification of their molecular structure. In the search for strategies to cope with the deleterious effect of mutagens, agents appeared which may reduce or eliminate such damage, the antimutagens. The basis for studying these substances is the knowledge that carcinogenesis is strongly related with mutagenesis, evidence supporting that carcinogenesis is highly due to the activity of environmental agents, and also, information that genotoxic inhibitors may frequently be found in plants and their products, as well as in other components of the diet; factors which favor their use under the appropriate conditions (Weisburger, 2001). In this context, it is pertinent to refer to Ames (1983) who suggested that a diet insufficient in fruits and vegetables may double the risk of acquiring cancer and cardiac diseases. This statement, as well as a number of reports on the matter increased the interest in determining the potency, toxicity, and mechanism of action of antimutagens as the necessary basis for incorporating the best candidates in preclinical and clinical chemoprevention trials. Moreover, studies on the matter have put into perspective the real beneficial action of this type of agents. Such studies have also considered various aspects that must be understood or solved, such as the fact that most antimutagens act on specific mutagens, and the possibility that the effect can be nullified or even reversed to mutagenicity in regard to the dose, time, and cell/organism tested, a possibility which is also complicated by the interactions that may occur between any compound and the complex human organism (De Flora & Ferguson, 2005; Ferguson, 2010).

The number of antimutagens and how they may act has been growing in recent years. Moreover, it is known that an agent may have more than one mechanism of action, and that two or more antimutagens could act synergistically. For more detailed information on the classification and the antimutagen's mechanism of action the reader may consult specific reports (De Flora et al., 2001; De Flora & Ferguson, 2005); however, for the sake of simplicity, in this revision antimutagens have been classified as desmutagens, impeding or limiting the effect of the mutagen before reaching the DNA molecule, such as the adsorbents that may interfere with the cellular absortion, or as those that avoid or reduce mutagen formation by blocking the biotransformation of premutagens through the inhibition of their

managing coal tar. This effect was experimentally confirmed by Yamagiwa and Ichikawa (Weisburger, 2001) in exposed rabbits. Later, with the identification of the DNA structure and the development of new analytical methods, carcinogenesis was clearly shown to be related with alterations in this molecule. Such a relation was confirmed in the 60s, determining the effect exerted by metabolites of specific carcinogens on the structure and function of DNA and studying the activity of carcinogens on various cellular systems. In this context, the term genotoxic was then used for identifying carcinogens acting on DNA, in contrast with others whose effect was exerted through other routes (Weisburger, 2001). At this time, it was evident that the presence of numerous physical, chemical, and biological genotoxic agents could affect human health, and also that these genotoxicants may be present in a wide range of human activities, such as those related with work, food, health or personal habits. This produced a sort of genotoxic saturation, a condition which stimulated a search for knowledge about the agent's molecular, cellular, and metabolic peculiarities as well as detailed characteristics of the xenobiotic-DNA interaction; furthermore, the consequences of such an interaction were investigated, particularly with

A number of educational recommendations were then suggested in order to counteract the genotoxic effects, in addition to establishing regulatory measures related to permissible limits for specific substances; besides, efforts were made for substituting genotoxic drugs with less dangerous ones, including appropriate modification of their molecular structure. In the search for strategies to cope with the deleterious effect of mutagens, agents appeared which may reduce or eliminate such damage, the antimutagens. The basis for studying these substances is the knowledge that carcinogenesis is strongly related with mutagenesis, evidence supporting that carcinogenesis is highly due to the activity of environmental agents, and also, information that genotoxic inhibitors may frequently be found in plants and their products, as well as in other components of the diet; factors which favor their use under the appropriate conditions (Weisburger, 2001). In this context, it is pertinent to refer to Ames (1983) who suggested that a diet insufficient in fruits and vegetables may double the risk of acquiring cancer and cardiac diseases. This statement, as well as a number of reports on the matter increased the interest in determining the potency, toxicity, and mechanism of action of antimutagens as the necessary basis for incorporating the best candidates in preclinical and clinical chemoprevention trials. Moreover, studies on the matter have put into perspective the real beneficial action of this type of agents. Such studies have also considered various aspects that must be understood or solved, such as the fact that most antimutagens act on specific mutagens, and the possibility that the effect can be nullified or even reversed to mutagenicity in regard to the dose, time, and cell/organism tested, a possibility which is also complicated by the interactions that may occur between any compound and the complex human organism (De Flora & Ferguson, 2005;

The number of antimutagens and how they may act has been growing in recent years. Moreover, it is known that an agent may have more than one mechanism of action, and that two or more antimutagens could act synergistically. For more detailed information on the classification and the antimutagen's mechanism of action the reader may consult specific reports (De Flora et al., 2001; De Flora & Ferguson, 2005); however, for the sake of simplicity, in this revision antimutagens have been classified as desmutagens, impeding or limiting the effect of the mutagen before reaching the DNA molecule, such as the adsorbents that may interfere with the cellular absortion, or as those that avoid or reduce mutagen formation by blocking the biotransformation of premutagens through the inhibition of their

reference to human health.

Ferguson, 2010).

activation at the cytochrome P-450. The other class of antimutagens corresponds to the bioantimutagens agents which may even reduce the level of mutations after the DNA has been damaged. To this group belong sequesters of mutagens and free radicals, agents that enhance the activity of phase II enzymes and the repair system, or those that reduce errors at the DNA replication level (Kada & Shimoi, 1987).

The term chemoprevention was coined by Sporn in the mid 70s, and during the following years it was defined as a procedure for the prevention, inhibition, delay, or reversal of carcinogenesis by means of a variety of agents which include different nutrients, extracts of plants or pharmacologic compounds, among others. The aim of the strategy refers to finding agents with several characteristics: 1-low cost, as related to cost-benefit analyses and to the size of the target population; 2- practicality of use, regarding availability, storage conditions and administration route, besides taking into account the need to be used for long periods of time; 3-efficacy; and 4-safety. The selected chemopreventive agents should protect target molecules, cells, general population and individuals at risk, against the initiation, promotion or progression phases of carcinogenesis (De Flora & Ferguson, 2005). The concept is based in that chronic diseases may have common pathogenic determinants, such as DNA damage, oxidative stress, and chronic inflammation, and that a number of agents have proved to be efficient in blocking such alterations and in improving the quality and span of human life. The more promising candidates are subjected to clinical trials, which should be designed and conducted properly, and should include well characterized agents, suitable cohorts, and reliable biomarkers for measuring efficacy, which can serve as surrogate endpoints for cancer incidence. Phase II chemoprevention trials test promising agents for biomarkers modulation in cohorts of 30 to 200 participants at greater than average risk of the cancer under study; in contrast, phase III trials test agents for their efficacy in cancer prevention in thousands of participants who are generally healthy or who may be at slightly elevated risk (Kelloff et al., 1995; Richmond & O´Mara, 2010).

#### **5. Genotoxicity/antigenotoxicity tests**

Genotoxicity/antigenotoxicity tests can be defined as in vitro and in vivo assays designed to detect both, compounds that induce genetic damage by various mechanisms, as well as those that prevent such damage. These tests enable hazard identification with respect to DNA damage and its fixation in the form of gene mutations, chromosomal aberrations or other alterations, all of which are considered essential for heritable effects and for the multistep process of malignancy (Figure 3). In contrast, the same tests may provide information on the level of protection and the mechanism involved regarding the antigenotoxic agents (Food and Drug Administration [FDA], 2008). In this section we will briefly describe the more basic fundaments regarding some of the tests most used to evaluate the prevention of DNA damage induced by AFB1.

a. The Ames mutation assay, which was developed in *Salmonella typhimurium* in the mid 70s, is based in the use of strains with a mutation in the histidine locus which does not allow the bacteria to synthesize such aminoacid; thereby, reversion to the normal situation constitutes the mutagenic endpoint. The sensitivity of the test has been improved by incorporating mutations to the test organism, making it more permeable to chemicals and more resistant to DNA repair (Dearfield & Moore, 2005). Moreover, several strains which detect different base-pair substitutions have been constructed, thereby allowing the detection of oxidative damage or DNA cross-linking. Besides,

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 261

d. Micronuclei are genotoxic lesions that may originate from acentric chromosome fragments or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell divisions (Organisation for Economic Co-operation and Development [OECD], 2007). The test can be designed to detect the activity of chemicals with clastogenic and aneugenic potential, in cells that have undergone cell divisions during or after exposure to the test substance. Because of easier performance and microscopic detection, as well as high sensitivity for detecting mutagens/antimutagens, it has partially substituted the chromosomal aberration analysis. The assay can be made in vitro and in vivo, as well as in animals or plants. In cultured cells, the incorporation of cytochalasin, a cytokinesis blocker that allows micronuclei evaluation in synchronic binucleated cells, has become popular. In mouse in particular, micronuclei can be scored in both immature polychromatic erythrocytes or mature, normochromatic erythrocytes. Moreover, the test can be applied for examining exfoliated cells, and its sensitivity can be increased by means of flow cytometry which allows the analysis as many as 500000 events (Dertinger et al, 2011;

e. The single cell gel electrophoresis assay, also known as comet assay, is a sensitive technique for detecting and analyzing DNA breakage in a variety of organs and various plant and animal cells. The basic principle resides in the migration of DNA in an agarose matrix under electrophoretic conditions, which depends on the level of breakage. When viewed under a microscope, a cell may have the appearance of a comet, with a head (the nuclear region) and a tail containing DNA fragments which have migrated in the direction of the anode. The length and the frequency of comets depend on the genotoxic potential of the tested agent (Oshida et al., 2008). The advantage of the comet assay is that it allows any viable eukariote cell to be analyzed for DNA damage, by detecting single or double strand breaks, alkali-labile sites that are expressed as single-strand breaks, and single-strand breaks associated with incomplete excision repair. Quantitative analysis for DNA damage has yielded several parameters, including tailed nuclei, tail length, percentage of DNA in the tail, and tail moment; besides, specific enzymes can be added to the test to analyze oxidative damage or the test can be integrated with the FISH assay for evaluating specific gene

position/movement (Hartmann et al., 2003; Kumaravel et al., 2009).

f. An adduct corresponds to a stable complex formed when a chemical is covalently linked to a macromolecule, such as protein or DNA. The measurement of adducts in

specifically identify cells in the first cellular division.

Fenech et al., 2011).

usually classified as chromosome and chromatid lesions, and more specifically, to deletions and fragments as well as various forms of chromosome intrachanges and interchanges. The studied cells and/or organisms can be selected based on their growth ability, stability of the karyotype, chromosome number, and spontaneous frequency of alterations (US Enviromental Protection Agency [EPA], 1998; Organisation for Economic Co-operation and Development [OECD], 2010). The in vivo or in vitro assay requires proliferating cells which are treated with the tested substance during an appropriate exposure time, and at the end, the test requires a hypotonic treatment, followed by the cell fixation and staining. Also, evaluating chromosomal aberrations in an initial round of cellular proliferation is important in order to have precise quantification of aberrations due to possible losses in subsequent divisions; therefore, the differential staining procedure applied to determine SCE can also be used to

limitation of the bacteria in regard to the absence of metabolic activation by enzymes common to the mammalian metabolism was overcome by adding rat liver microsome homogenate (S9 homogenate) to the bacterial cultures (Ames et al., 1973; Dearfield & Moore, 2005).

b. SCE represent the interchange of DNA replication products at apparently homologous chromosomal loci; such exchanges presumably involve DNA breakage and reunion (Latt & Schreck, 1980). The test can be made in vitro or in vivo. In the first case a number of cellular lines or primary cultures from different organisms can be used, with or without the addition of S9 for inducing metabolic activation. Also, a main step in making the test is to differentially stain the sister chromatids in such a way that they can be clearly visualized as a distinct chromatid in second division metaphases; this is essential for counting the number SCE per chromosome/cell, which is the evaluated genotoxic endpoint. The compound bromodeoxyuridine is usually added to the cultures or intraperitoneally injected to the test animal to visualize the sister chromatids; this compound is a thymidine analogue which is readily incorporated into the DNA chains and acts as a molecular marker during DNA replication, which is reflected as the differentially stained chromatids when colored with a Giemsa stain (Latt & Schreck, 1980).

Fig. 3. Examples of genotoxic lesions. A) DNA damage observed with the comet assay, B) Sister cromatid exchanges in mouse bone marrow cells, C) micronuclei in mouse erythrocyte, D) DNA adduct (8-hydroxy-2´-deoxiguanosine)

c. Chromosomal aberrations are generally classified under two types: a numerical one and a structural one, and although both types are useful for the analysis of genotoxicity, the structural type of anomalies, is probably the more utilized. This type of aberrations is

b. SCE represent the interchange of DNA replication products at apparently homologous chromosomal loci; such exchanges presumably involve DNA breakage and reunion (Latt & Schreck, 1980). The test can be made in vitro or in vivo. In the first case a number of cellular lines or primary cultures from different organisms can be used, with or without the addition of S9 for inducing metabolic activation. Also, a main step in making the test is to differentially stain the sister chromatids in such a way that they can be clearly visualized as a distinct chromatid in second division metaphases; this is essential for counting the number SCE per chromosome/cell, which is the evaluated genotoxic endpoint. The compound bromodeoxyuridine is usually added to the cultures or intraperitoneally injected to the test animal to visualize the sister chromatids; this compound is a thymidine analogue which is readily incorporated into the DNA chains and acts as a molecular marker during DNA replication, which is reflected as the differentially stained chromatids when colored with a Giemsa stain

Fig. 3. Examples of genotoxic lesions. A) DNA damage observed with the comet assay, B)

D

B

c. Chromosomal aberrations are generally classified under two types: a numerical one and a structural one, and although both types are useful for the analysis of genotoxicity, the structural type of anomalies, is probably the more utilized. This type of aberrations is

Sister cromatid exchanges in mouse bone marrow cells, C) micronuclei in mouse

erythrocyte, D) DNA adduct (8-hydroxy-2´-deoxiguanosine)

Moore, 2005).

(Latt & Schreck, 1980).

A

C

limitation of the bacteria in regard to the absence of metabolic activation by enzymes common to the mammalian metabolism was overcome by adding rat liver microsome homogenate (S9 homogenate) to the bacterial cultures (Ames et al., 1973; Dearfield & usually classified as chromosome and chromatid lesions, and more specifically, to deletions and fragments as well as various forms of chromosome intrachanges and interchanges. The studied cells and/or organisms can be selected based on their growth ability, stability of the karyotype, chromosome number, and spontaneous frequency of alterations (US Enviromental Protection Agency [EPA], 1998; Organisation for Economic Co-operation and Development [OECD], 2010). The in vivo or in vitro assay requires proliferating cells which are treated with the tested substance during an appropriate exposure time, and at the end, the test requires a hypotonic treatment, followed by the cell fixation and staining. Also, evaluating chromosomal aberrations in an initial round of cellular proliferation is important in order to have precise quantification of aberrations due to possible losses in subsequent divisions; therefore, the differential staining procedure applied to determine SCE can also be used to specifically identify cells in the first cellular division.


Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 263

mechanism of action, a strong complex formation between mutagen and antimutagen was

Based on the described positive experimental studies, efforts were initiated to determine the CHL chemopreventive capacity in humans. Qidong, People's Republic of China is a high risk region for hepatocellular carcinoma probably related with the consumption of AFB1 contaminated food; here, a randomized, double-blind, placebo-controlled trial was made to determine whether CHL administration altered the disposition of aflatoxin. CHL consumption at each meal led to an overall 55 % reduction in a median urinary level of the biomarker, aflatoxin-N7-guanine, compared with individuals taking placebo (Egner et al., 2001). The determined adduct biomarker derives from the carcinogenic metabolite, aflatoxin 8,9-epoxide; thus, the authors suggested that prophylactic interventions with CHL or supplementation of diets with chlorophyll rich foods may be useful to prevent the development of hepatocellular carcinoma or other environmentally induced cancers. This type of studies was supported by reports on the experimental effect of CHL in rats (Simonovich et al., 2007). The authors observed the inhibition of AFB1-albumin adducts and of AFB1-N7-guanine adducts, as well as the inhibition of AFB1 uptake when quantified in feces, besides a decrease in the number of colonic aberrant crypt foci induced by the aflatoxin. However, no modification in the activity of phase II enzymes

In summary, a number of in vitro and in vivo studies have supported the antigenotoxic and chemopreventive capacity of CHL against the damage induced by AFB1, activities which can be related with the formation of a strong non-covalent complex, although additional mechanisms, such as its antioxidant potential, cannot be discarded. However, a word of caution about safety in using CHL is pertinent in light of the negative or controversial results that have been published in regard to the compound: for example, its effect as both inhibitor or promoter of genetic damage depending on the tested approach (Cruces et al.,

proposed.

was found.

Fig. 4. Chemical structure of chlorophyllin

body fluids is highly sensitive and specific to determine the effect of the studied xenobiotic. DNA adducts have been clearly shown to be relevant to the disease process in prospective studies (Bonassi & Au, 2002). In relation to our present review, the adducts 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin, as well as the AFB1 formamidopyrimidine compound, among others, are thought to be involved in the mutations caused by AFB1. The detection of these compounds can be made in various organs, as well as in serum and urine, by means of a variety of methods that include HPLC, ELISA, accelerator mass spectrometry, and liquid chromatography/electrospray ionization/mass spectrometry (Sharma & Farmer, 2004; Wang et al., 2008).
